There has been a steady increase in the incidence of fungal infections over the past few decades, primarily due to the AIDS pandemic. Candida albicans is the most common human fungal pathogen, causing both mucosal and systemic infections and remains a major clinical problem primarily in immunocompromised patients. It has been reported that 60 to 80% of HIV infected patients develop one or more fungal infections at some time during their illness, the most frequent one being oropharyngeal candidiasis (1). Candidiasis caused by Candida spp is the fourth most common cause of nosocomial infectious disease (NID) in the US, and there is a similar trend world-wide (2-7).The magnitude of NID caused by fungi is reflected in patient costs. According to one estimate, during 1998, the cost of treatment of invasive fungal infections, estimated to be approximately 62,000 cases per year in the US, was $2.6 billion, and the average per-patient attributable cost was $31,200 (5). Given the clinical importance of candidiasis, there is an urgent need to identify new drug targets and therefore new anti-fungal drugs, to improve diagnostic assays and to provide alternative therapeutic options such as the use of passive immune anti-Candida antibodies or vaccines for the treatment of high-risk patients. However, since like humans, fungi are eukaryotes, targets for anti-fungal drug discovery must not be found in humans and, in addition, present in all fungal pathogens. Two-component signal transduction proteins have been reported in bacteria and lower eukaryotes. To date they have not yet been identified in animals and are absent in the human genome. Previous studies have confirmed the role of the two-component signal proteins in the pathogenesis of C. albicans in a mouse model of hematogenously disseminated candidiasis, survival in human PMNs in vitro, adherence to human esophageal tissue, quorum sensing and adaptation to oxidants. These features make two-component proteins very attractive targets for antifungal drug discovery since they have important functions and are not found in humans. Two-component phosphorelay systems include a membrane-bound, sensor histidine kinase (HK) protein, which in bacteria, is autophosphorylated in response to an environmental stimulus. Phosphotransfer to a cytoplasmic response regulator (RR) protein occurs which in turn transmits the signal, either through direct transcriptional activation of target genes, or by activation of a down stream mitogen-activated protein kinase cascade (MAPK). The genome of Candida albicans includes three HKs and two RRs, of which Ssk1p is a response regulator that is not functionally related to the Saccharomyces cerevisiae homologue. I have tested two-component and downstream MAP kinase mutants for sensitivity to available antifungal drugs. My preliminary results of MIC broth micro dilution assays indicate that the ssk1 mutant is hypersensitive to the triazoles, fluconazole and voriconazole compared to the wild type strain CAF2-1. Interestingly, the ssk1 mutant retains the wild type levels of sensitivity to other antifungals such as miconazole and ketoconazole (imidazoles), amphotericin B, caspofungin, 5-FC, and a variety of other inhibitors such as Congo red, calcofluor white, hygromycin and nikkomycin Z. So the hypersensitivity of the ssk1 mutant to triazoles appears to be very specific. Further, new data indicate that the ssk1 mutant has elevated levels of fluconazole uptake and lost viability upon incubation with fluconazole and voriconazole, suggesting that in the absence of Ssk1p, fluconazole and voriconazole have significantly increased fungicidal effect on C. albicans. I have also identified several transport proteins by microarray analysis that are up-regulated in ssk1 mutant. Based on this preliminary data, I hypothesize that the Ssk1 protein is a promising therapeutic target and may be exploited in the development or identification of new antifungal drugs. Equally important, my studies will provide new observations on the uptake of triazoles, of which there is very little information in the literature. I also hypothesize that therapy which includes a triazole and a drug which targets Ssk1p or proteins it regulates may enable triazoles to be fungicidal rather than fungistatic. There are two specific aims in this research proposal. In Specific aim 1, part A, I will characterize fluconazole uptake in the ssk1 mutant compared to wt cells in order to explain the hypersensitivity of the mutant. In part B, I will identify the transporter(s) responsible for fluconazole uptake. In Specific aim 2, post antibiotic effects (PAE) will be analyzed to resolve the question as to whether the hypersensitivity of the ssk1 mutant to triazoles also occurs following removal of triazoles. This phenomenon notoriously results in persistence of the organism in the patient as the drug concentrations decrease. In part A of specific aim 2, the PAE of wt and mutant cells will be compared. In part B, gene transcription of wt and the ssk1 mutant at time points during exposure of the drugs and post-exposure will be examined. Recent data by another lab indicate that molecular events can be predicted during post-exposure that indicate recovery of the organism. As part of this sub aim, I will also begin to analyze downstream effector proteins by transcriptional profiling to identify genes regulated by SSK1 that are associated with the hypersensitivity phenotype. Long-term objectives of this research will focus upon the development of high throughput assays to identify compounds that inhibit Ssk1p or downstream proteins that are regulated by Ssk1p. PUBLIC HEALTH RELEVANCE There has been a steady increase in the incidence of fungal infections over the past few decades, primarily due to the AIDS pandemic. Candida albicans is the most common human fungal pathogen, causing both mucosal and systemic infections and remains a major clinical problem primarily in immunocompromised patients. It has been reported that 60 to 80% HIV infected patients develop one or more fungal infection at some time during their illness, the most frequent one being oropharyngeal candidiasis (1). Candidiasis caused by Candida spp is the fourth most common cause of nosocomial infectious disease (NID) in the US, and there is a similar trend world-wide (2-7).The magnitude of NID caused by fungi is reflected in patient costs. According to one estimate, during 1998, the cost of treatment of invasive fungal infections, estimated to be approximately 62,000 cases per year in the US, was $2.6 billion, and the average per-patient attributable cost was $31,200 (5). The present study is aimed at the development and identification of newer antifungal drug targets which may be exploited in anti-fungal drug discovery. [unreadable] [unreadable] [unreadable]